Cell penetrating peptides (CPPs) are able to transport hydrophilic molecules inside cells. To reach the cytosol, the peptide associated with a cargo must cross the plasma or the endosomal membrane. Different molecular mechanisms for peptide internalisation into cells have been proposed and it is becoming clear that the cellular internalisation mechanisms are different depending on the peptide sequence and structure and the target membrane. Herein, the penetration of three peptides into large unilamellar vesicles were studied: the homeodomain derived 16-residues penetratin, nona-arginine (R9), and a small peptide containing 6 arginine and 3 tryptophan residues (RW9). The membrane models were composed of phospholipids from natural sources containing different molecular species. We observed that among the three peptides, only the amphipathic peptide RW9 was able to cross the membrane vesicles in the liquid disordered state. The changes in the distribution of the previously characterized cholesterol-pyrene probe show that cholesterol-pyrene molecules dissociate from clusters upon membrane interaction with the three peptides and that the cholesterol environment becomes more disordered in the presence of RW9. Finally, we studied the effect of the peptides on lipid ordering on giant plasma membrane vesicles. The amphipathic peptides RW9 and its longer homologue RW16 induced lipid de-packing in plasma membrane vesicles. Overall, the data suggest that a disordered membrane favours the translocation of RW9, that the membrane cholesterol is redistributed during peptide interaction, and that the peptide amphipathic character is important to increase membrane fluidity and peptide membrane translocation.